Efficient and Stable Transgene Expression in Human Embryonic Stem Cells Using Transposon-Mediated Gene Transfer

Wilber, Andrew; Linehan, Jonathan L.; Tian, Xu; Woll, Petter S.; Morris, Julie; Belur, Lalitha R.; McIvor, R. Scott; Kaufman, Dan S.

doi:10.1634/stemcells.2007-0026

Cited by 107 publications

(94 citation statements)

References 51 publications

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“…However, this system requires adaptation for hESCs and application of effective human promoters rather than the CMV promoter [48,49]. Also, new nonviral transposon-mediated gene transfer systems may be a safer option [50], also promising high efficiency and inducibility in embryonic stem cells [51,52]. Generally, HoxB4 regulation could avoid possible side effects caused by expression above the therapeutic level.…”

Section: Discussionmentioning

confidence: 99%

Lentiviral-Mediated HoxB4 Expression in Human Embryonic Stem Cells Initiates Early Hematopoiesis in a Dose-Dependent Manner but Does Not Promote Myeloid Differentiation

et al. 2008

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The variation of HoxB4 expression levels might be a key regulatory mechanism in the differentiation of human embryonic stem cell (hESC)-derived hematopoietic stem cells (HSCs). In this study, hESCs ectopically expressing high and low levels of HoxB4 were obtained using lentiviral gene transfer. Quantification throughout differentiation revealed a steady increase in transcription levels from our constructs. The effects of the two expression levels of HoxB4 were compared regarding the differentiation potential into HSCs. High levels of HoxB4 expression correlated to an improved yield of cells expressing CD34, CD38, the stem cell leukemia gene, and vascular epitheliumcadherin. However, no improvement in myeloid cell maturation was observed, as determined by colony formation assays. In contrast, hESCs with low HoxB4 levels did not show any elevated hematopoietic development. In addition, we found that the total population of HoxB4-expressing cells, on both levels, decreased in developing embryoid bodies. Notably, a high HoxB4 expression in hESCs also seemed to interfere with the formation of germ layers after xenografting into immunodeficient mice. These data suggest that HoxB4-induced effects on hESC-derived HSCs are concentrationdependent during in vitro development and reduce proliferation of other cell types in vitro and in vivo. The application of the transcription factor HoxB4 during early hematopoiesis from hESCs might provide new means for regenerative medicine, allowing efficient differentiation and engraftment of genetically modified hESC clones. Our study highlights the importance of HoxB4 dosage and points to the need for experimental systems allowing controlled gene expression.

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Section: Discussionmentioning

confidence: 99%

Lentiviral-Mediated HoxB4 Expression in Human Embryonic Stem Cells Initiates Early Hematopoiesis in a Dose-Dependent Manner but Does Not Promote Myeloid Differentiation

et al. 2008

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“…Thirty days after nucleofection, more than 95% of cells demonstrated GFP expression by flow cytometry with luciferase expression of 957 RLU/ 15 seconds per cell by enzymatic assay as previously described. 19 …”

Section: Generation Of Luc ؉ K562 Cellsmentioning

confidence: 99%

“…This model allows serial bioluminescent imaging to noninvasively monitor growth or regression of the tumor cells in individual animals over a prolonged time course. 21 To avoid viral transfection that could make K562 cells more susceptible to NK cell-mediated lysis, 19,22 we chose to generate luc ϩ cells by Sleeping Beauty transposon-mediated transfection as described in "Methods" in "Generation of luc ϩ K562 cells" ( Figure S1, available on the Blood website; see the Supplemental Materials link at the top of the online article). Luc ϩ K562 cells were injected subcutaneously into sublethally irradiated NOD/SCID mice and allowed to grow for 3 days to provide an established tumor before NK-cell immunotherapy.…”

Section: Increased In Vivo Antitumor Activity Of Hesc-nk Cells Comparmentioning

confidence: 99%

Human embryonic stem cells differentiate into a homogeneous population of natural killer cells with potent in vivo antitumor activity

Woll

Grzywacz

Tian

et al. 2009

Blood

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226

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“…In difficult-to-transfect cells, including primary stem cells, delivery of transposon-based vectors can be significantly facilitated by nucleofection, a procedure based on electroporation that transfers nucleic acids directly into the nucleus. Indeed, nucleofection facilitated transposition in various stem cells including CD34 + hematopoietic progenitors (Hollis et al, 2006;Izsvák et al, 2009;Mates et al, 2009;Sumiyoshi et al, 2009;Xue et al, 2009), primary T cells (Huang et al, 2008Singh et al, 2008) and human embryonic stem cells (Wilber et al, 2007;Orban et al, 2009). Importantly, in the context of the hematopoietic system, this ex vivo gene delivery procedure apparently did not compromise the potential of transposon-marked CD34 + cells to differentiate normally into the erythroid, megakaryocytic, granulocyte/monocyte/macrophage lineage as well as into the CD4 + CD8 + T, CD19 + B, CD56 + CD3 -natural killer (NK), and CD33 + myeloid lineages (Xue et al, 2009).…”

Section: Delivery Of the Sleeping Beauty Vector Systemmentioning

confidence: 99%

Nonviral Gene Delivery with the Sleeping Beauty Transposon System

Ivics

Izsvák²

2011

Human Gene Therapy

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Effective gene therapy requires robust delivery of therapeutic genes into relevant target cells, long-term gene expression, and minimal risks of secondary effects. Nonviral gene transfer approaches typically result in only short-lived transgene expression in primary cells, because of the lack of nuclear maintenance of the vector over several rounds of cell division. The development of efficient and safe nonviral vectors armed with an integrating feature would thus greatly facilitate clinical gene therapy studies. The latest generation transposon technology based on the Sleeping Beauty (SB) transposon may potentially overcome some of these limitations. SB was shown to provide efficient stable gene transfer and sustained transgene expression in primary cell types, including human hematopoietic progenitors, mesenchymal stem cells, muscle stem/progenitor cells (myoblasts), induced pluripotent stem cells, and T cells. These cells are relevant targets for stem cell biology, regenerative medicine, and gene-and cell-based therapies of complex genetic diseases. Moreover, the first-in-human clinical trial has been launched to use redirected T cells engineered with SB for gene therapy of B cell lymphoma. We discuss aspects of cellular delivery of the SB transposon system, transgene expression provided by integrated transposon vectors, target site selection of the transposon vectors, and potential risks associated with random genomic insertion.

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Efficient and Stable Transgene Expression in Human Embryonic Stem Cells Using Transposon-Mediated Gene Transfer

Abstract: ABSTRACT

Cited by 107 publications

References 51 publications

Lentiviral-Mediated HoxB4 Expression in Human Embryonic Stem Cells Initiates Early Hematopoiesis in a Dose-Dependent Manner but Does Not Promote Myeloid Differentiation

Lentiviral-Mediated HoxB4 Expression in Human Embryonic Stem Cells Initiates Early Hematopoiesis in a Dose-Dependent Manner but Does Not Promote Myeloid Differentiation

Human embryonic stem cells differentiate into a homogeneous population of natural killer cells with potent in vivo antitumor activity

Nonviral Gene Delivery with the Sleeping Beauty Transposon System

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